Generating cardiovascular cells from pluripotent stem cells holds great promise for cardiovascular research and therapy. However, pluripotent stem cell differentiation into cardiac cells is inefficient and results in heterogeneous cultures, limiting the usefulness of this approach. Pluripotent stem cells, such as human embryonic stem (hES) cells and induced pluripotent stem (iPS) cells, can perpetually proliferate and differentiate into derivatives of all three embryonic germ layers (Thomson et al., Science 282:1145 (1998); Odorico et al., Stem Cells 19:193 (2001); Yu et al., Science 318(5858):1917 (2007)). Differentiation of pluripotent stem cell cultures can occur spontaneously, which results in a seemingly random variety of cells (Watt and Hogan, Science 287:1427 (2000)). The earliest methods of pluripotent stem cell differentiation included allowing stem cell aggregates to spontaneously differentiate and form embryoid bodies (EBs) which contain derivatives of the three primary germ layers including in some cases cardiomyocytes.
Generating cardiomyocytes from pluripotent stem cells through EB formation is inefficient, however, as only few percent of the developing cells become cardiomyocytes. Efficient, reproducible methods for differentiating human pluripotent stem cells into cardiovascular cell lineage remain to be elucidated.
More recently, researchers attempted to improve efficiency by differentiating hES cells into cardiomyocytes without EB formation by sequentially applying growth factors or small molecules to mimic cardiac development. Soluble factors important for embryonic cardiac development include Activin A, BMP4, nodal, Wnt agonists and antagonists, bFGF and other molecules (Conlon et al., Development 120(7):1919 (1994); Lough et al., Dev. Biol. 178(1):198 (1996); Mima et al., PNAS 92(2):467 (1995); Zaffran and Frasch, Circ. Res. 91 (6), 457 (2002)). Various combinations of these factors have been tested in cardiac differentiation protocols. Treatment with Activin A and BMP4 promoted cardiogenesis of H7 ES cells grown as monolayers (Laflamme et al., Nat. Biotechnol. 25 (9):1015 (2007)). Other protocols employed EB-based differentiation and enhanced cardiogenesis by using various combinations of BMP4, Activin A, bFGF, VEGF, and dickkopf homolog 1 (DKK1) (Yang et al., Nature 453 (7194):524 (2008)). The latter protocols were performed using H1 and HES2 human ES cell lines and have not been demonstrated to work with other ES cell lines or iPS cell lines.
iPS cells are generated by reprogramming somatic cells or differentiated progenitor cells to a state of pluripotency. Apart from their somatic cell origin, iPS cells share many characteristics of embryonic stem cells, such as the ability to grow perpetually and to differentiate into cells of all three germ layers. Like ES cells, iPS cells express pluripotency markers, such as OCT-4, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, and Nanog. iPS cells have been generated using retroviral vectors that randomly insert exogenous DNA into the target cell genome. More recently, vector- and transgene-free iPS cells have been generated by using non-integrating vectors. Using non-integrating vectors avoids the risk of aberrant cellular gene expression and neoplastic growth (Okita et al. Nature 448:313 (2007)). Loss of the reprogramming vector also avoids perpetual expression of transgenes that can induce programmed cell death (apoptosis) (Askew et al., Oncogene 6:1915 (1991), Evan et al., Cell 69:119 (1992)) and interfere with subsequent differentiation of iPS cells.
More recently, methods were devised for reprogramming somatic cells using oriP/Epstein-Barr nuclear antigen-1 (EBNA1)-based episomal vectors that do not integrate into the genome and are lost from the cells after reprogramming (Yu et al., Science 324(5928):797 (2009)). iPS cells generated by this method are vector- and transgene-free and, as such, are well suited for clinical application. However, the ability of these vector-free iPS cells to form EBs or to differentiate into various cells types, such as cardiomyocytes, useful for clinical application has been unknown.
During embryonic development as the primitive streak forms, a migratory population of the epiblast cells undergoes an epithelial-to-mesenchymal transition (EMT) during gastrulation to generate mesodermal cells. This marks the first step in differentiation to mesoderm and, ultimately, to cells of the mesodermal lineage, such as cardiomyocytes.
EMT is an important biological phenomena implicated in multiple steps of development and other healthy and diseased physiological processes, such as cancer metastasis. For example, breast cancer cells initially are characterized by an epithelial phenotype but assume a mesenchymal phenotype during tumor metastasis. This transformation enables the cells to dissociate from their tissue of origin, enter the circulation, and establish metastases in remote tissues sites.
Prior to the inventors' work, no efficient cardiac differentiation protocol demonstrated broad applicability across multiple ES and iPS cell lines. Also, available differentiation protocols relied on the application of soluble growth factors to direct differentiation and failed to consider the role of extracellular matrix in promoting the first differentiation steps of ES and iPS cells necessary to generate mesoderm and subsequent cardiomyocytes.